JPH0646942B2 - New Bulgarian lactobacillus - Google Patents

Description

Detailed Description of the Invention

The present invention relates to a novel lactic acid bacterium of Bulgaria.

Fermentative production of D-lactic acid is described in British Patent No. 115.
It is known from the description of the 7213 specification. Lactobacillus (Lactobacil) used in this method
lusleichmanii) ATCC 4797 can produce 113 g of D-lactic acid per liter of nutrient medium containing glucose or beet molasses alongside other conventional additives within 60 hours.

Due to the increasing demand for D-lactic acid as a starting material for the chemical synthesis of optically active compounds, it is an object of the present invention to develop an efficient method for obtaining it. In that case, in particular, it is desirable to reduce the fermentation time and to find not only glucose and sugar beet molasses but also microorganisms that utilize lactose which are available in large quantities in whey. This purpose is Lactobacillus bulgaric
us).

The lactic acid bacterium Bulgarian lactobacillus is known to be able to ferment particularly rapidly due to its high metabolic activity. It is therefore used in the preparation of yogurt and can complete milk fermentation within 4 hours. However, the lactobacillus strain of Bulgaria present in natural milk and fermented milk cannot produce more than 25-30 g of lactic acid per liter of medium.
More than half of the lactose present remains unfermented.

Surprisingly, in an acidified milk sample, its efficiency is now 115 g / l of culture medium due to the systematic selection.
A strain of Lactobacillus bulgaria was found that could be improved to produce up to lactic acid. Accurate identification of this strain showed that it consisted of a long Gram-positive bacillus that was catalase-negative, non-motile and microaerobic and had no spores. In addition, it features the following data:

[0006]

This strain is a German microorganism depository (Deutsc
he Sammelung fuer Mikroorganismen) to DSM 212
Deposited as 9.

[0008] This new Bulgarian lactobacillus strain is 48
Within hours, up to 115 g of D-lactic acid can be produced per liter of medium. Furthermore, this is not specific to glucose fermentation,
Lactose can be fermented to lactic acid as quickly as glucose. Therefore, whey, which is available in large quantities as dairy waste, can be used as a source material source for producing D-lactic acid.

The present invention therefore uses Lactobacillus bulgaria DSM 2129 as a lactic acid producing microorganism in the preparation of D-lactic acid by fermentation of a nutrient medium containing glucose and / or lactose and other conventional additives. It also relates to a method consisting of things. Lactose is then usually used in the form of a fresh whey or suspension of whey powder, which optionally may also have glucose added.

The nutrient medium used to obtain D-lactic acid is constructed in a manner known per se. They must, of course, also contain a nitrogen source such as meat extract, corn steep or soy flour alongside glucose and / or lactose. In addition, mineral salts, vitamins and surfactants are also added. Surfactants include in particular liquid products, especially commercial nonionic surfactants. These surfactants generally contain the reaction products of polyoxyalkyl chelates such as alcohols or acids with ethylene oxide and / or propylene oxide as active substances. Alcohols include monohydric and polyhydric alcohols such as aliphatic alcohols, resin alcohols, glycerin, erythritol, pentaerythritol or sugar alcohols (eg sorbitol and mannitol), and acids in particular fatty acids and resin acids. Preference is given to partial esters of such polyhydric alcohols with said acids, such as the oxyethylates of anhydrosorbitol monooleate.

Since Lactobacillus bulgaria DSM 2129 is acid-sensitive, the lactic acid formed must be combined with alkali or alkaline earth hydroxides or carbonates, especially calcium carbonate. Therefore, the pH value is kept in the range of 4.5-7, preferably 6.5-6.8.

[0012] Each nutrient medium contains lactic acid bacterium Bulgarian DSM 2129 in order to remove any contamination by external organic matter.
Should be sterilized before inoculation. For this, heating the nutrient medium to 121 ° C. for 15 minutes is sufficient.

Anaerobic conditions should be maintained for the process of the present invention as in all lactic acid fermentations. Carbon dioxide produced by neutralizing lactic acid with calcium carbonate or a nitrogen blanket on the fermentation medium is sufficient for this.

The lactic acid fermentation can be carried out in the temperature range of 30-50 ° C. A particularly convenient temperature range is 40-45 ° C.

Free D-lactic acid can be obtained from the salt of D-lactic acid obtained by the method of the present invention by ion exchange or, in the case of calcium lactate, acidification with sulfuric acid.

The invention will be explained in more detail by the following examples. % Is by weight.

Example 1 Development of Lactobacillus bulgaria DSM 2129 Strain The Lactobacillus bulgaria strain found in a sample of fermented milk was isolated and cultivated on medium 1 below (numbers are g / l).

Casein peptone (trypsin digestion) 10 Meat extract (Merck product) 10 Yeast extract 5 Glucose 20 K ₂ HPO ₄ 2 Sodium acetate 5 MgSO ₄ .7H ₂ O 0.2 MnSO ₄ .H ₂ O 0. 05 Nonionic surfactant 1ml / l

A sample of the strain is then diluted and 1.8%
Were inoculated on agar plates prepared from the same medium supplemented with. The inoculated agar plates were then stored under anaerobic conditions in an incubator at 45 ° C for 1 day.

A total of 30 individual colonies were then picked and added to the culture vials containing medium 4 below (numbers are g / l).

Glucose 50 CaCO ₃ 70 Yeast extract 7 Corn steep (dry matter) 15 Sodium acetate 5 Nonionic surfactant 1 ml / l

The culture time was 24 hours at 45 ° C. After 24 hours, differences in the amount of D-lactic acid formed in each of the various well-fermented cultures were clearly discerned. The vial with the highest D-lactic acid content was used for a new smear (incubation) on the agar plate. This process was repeated regularly for 8 weeks, when the sugar content of the medium was initially 8% and after another 5 weeks rose to 10%. This strain was subsequently selected for a further 12 weeks until it was finally able to metabolize 100 g of glucose per liter of medium up to 65% in 24 hours, forming 55 g of D-lactic acid per liter. Complete glucolysis was complete after 36-40 hours.

Example 2 The lactobacillus bulgaria strain obtained according to Example 1 was inoculated into a culture vial containing 15 ml each of Medium 1. The vials were placed vertically for culturing at 45 ° C. for at least 8 hours and up to 20 hours and then used as inoculum for the cultures in Erlenmeyer flasks containing 250 ml of medium 1 each. These were similarly stored at 45 ° C. for 8 to 20 hours.

Next, the contents of the above 6 Erlenmeyer flasks were used as an inoculum of a fermenter having an internal volume of 30 liters to which the following medium 2 (the number is g / l) was added in advance.

Glucose 30 CaCO ₃ 15 Yeast extract 7 Casein peptone 10 Corn steep (dry matter) 20 Sodium acetate 5 Nonionic surfactant 1 ml / l

The fermentor is agitated at 100 revolutions per minute and 4
After a lapse of 8 to 12 hours at 5 ° C., a content of 270 l containing the medium 4 described in Example 1 or the following medium 3 (the numbers are g / l)
Was added to the fermentor.

Glucose 30 CaCO ₃ 70 Yeast extract 7 Soybean flour 15 Sodium acetate 5 Nonionic surfactant 1 ml / l

After the start of growth of the culture, that is, after about 6 to 8 hours, further glucose was added. The amount of glucose to be added depends on glucose consumption and acid formation. Total glucose was added within 40-50 hours, which corresponded to about 10% by weight of the nutrient medium. From glucose, 110-115 g of D-lactic acid (corresponding to 159-166 g of calcium lactate) were formed per liter of culture medium. The isolation of the free lactic acid is carried out by known methods, namely by calcium precipitation with sulfuric acid, filtration, evaporation and purification by distillation as ethyl ester or methyl ester.

The use of medium 3 has the advantage that the final product does not contain any appreciable amount of L-lactic acid. In contrast, medium 4 has the advantage that it can be filtered significantly better than medium 3 after fermentation. This is because the soybean flour in Medium 3 is only partially decomposed. The product prepared with medium 4 contains 1.5-2% L-lactic acid from corn steep in the medium.

After filtration, the L-lactic acid portion can be decomposed by passing the filtrate through an enzymatic reactor containing an enzyme that specifically decomposes L-lactic acid. Systems using L-lactate oxidase, L-lactate dehydrogenase or cytochrome b ₂ are suitable. L-lactate dehydrogenase requires NAD as a cofactor,
It can likewise be bound to a carrier and then regenerated and used again. Hexacyanoferrate is a suitable cofactor for cytochrome b ₂ , which can be reoxidized at the noble metal electrode by an electric current.

Example 3 A strain culture and an inoculum were prepared by the procedure described in Example 2, except that the nutrient medium used was the following medium 5 containing lactose as a substrate to be fermented (the number is g / l). used.

Whey powder
60 CaCO ₃ 70 Yeast extract 3 Soybean flour 5 Sodium acetate 5 Nonionic surfactant 1 ml / l

As in Example 2, after the start of growth of the culture, the total amount of lactose added with the sugar substrate was 13 per liter of medium.
Added until 0 g. Lactic acid production lasted somewhat longer than in Example 2, but ended after 45-55 hours.
Others were operated in the same manner as in Example 2.

Example 4 Strain cultures and inoculums were prepared under a nitrogen blanket as in Example 1. Media 3, 4 or 5 were used, but they initially contained no CaCO ₃ . It is added later either in dry form or as a 20% suspension, so that the pH value never drops below 5.5. The addition of the nutrient solution was started after 48 hours. The dilution rate was initially 0.01 l / l of nutrient solution, with the same amount of liquid being continuously removed from the fermentor. The dilution rate is 0.03 to 0.04 per liter of nutrient solution per hour.
It was gradually increased to a value of 1 with the effluent from the fermentor exhibiting a D-lactic acid content of 5 to 7%. The cell mass was separated from the effluent using a continuous separator and 90% of it was returned to the fermentor. The supernatant was adjusted to pH 6.5 with calcium hydroxide and evaporated to about 1/10 of its original volume. When the concentrate was cooled to 4 ° C in a cold trap, calcium lactate crystallized out. The concentrated solution continuously flowed into the cold trap, while the precipitated calcium lactate was ejected at regular intervals by the slide valve. The mother liquor was recycled to the fermentor. Release of lactic acid from the obtained calcium lactate was accomplished by sulfuric acid and subsequent filtration.

Example 5 The continuous lactic acid preparation according to Example 4 was repeated except that sodium hydroxide was used instead of CaCO ₃ for pH adjustment. The advantage of this method is that the pH can be maintained at 6.5-6.8, which increases the fermentation rate of Lactobacillus bulgaria DSM 2129. The resulting solution of sodium lactate is added to the ion exchanger column that adsorbs lactic acid. As soon as the column is loaded with lactic acid, it is eluted with hydrochloric acid. It can be reused for adsorption of lactic acid after regeneration using column-diluted caustic soda.

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Claims (1)

[Claims] 1. Lactobacillus bulgaricus DSM-2129 having D-lactic acid producing ability.